Dentin defects caused by a Dspp−1 frameshift mutation are associated with the activation of autophagy

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5’ mutations affecting targeting and trafficking, and 3’ − 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp−1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp−1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp−1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp−1fs mice may be valuable for the study of autophagy and ER-phagy.


Results
Odontoblast pathosis in the Dspp −1fs mice. Odontoblast pathosis in homozygous Dspp −1fs continuously growing mandibular incisors was visualized at high resolution using focused ion beam scanning electron microscopy (FIB-SEM). For comparison, FIB-SEM of WT odontoblasts and dentin formation are provided (Figs. 1, S1). Dentin formation in WT mice showed well-aligned early odontoblasts developing their characteristic columnar and polarized morphology, with the nucleus positioned proximally ( Figure S1A). These odontoblasts showed abundant endoplasmic reticulum (ER), Golgi apparatus, secretory vesicles, and mitochondria (Figs S1B,C,D, 1A,B,C,D), indicative of functional secretory activity. Most of the ER cisternae were parallelly aligned in the odontoblasts, and mitochondria were consistently found nearby (Fig. 1a,c,d,e). Some ER cisternae with a clear boundary were enlarged (Fig. 1a), while some showed relatively rough borders (Fig. 1e). At the distal end, the odontoblastic processes extended from the cell bodies into the pre-dentin and dentin matrixes and branched into smaller processes, some of which almost extended to the DEJ (Fig. 1A,B,C and 1b). Collagen bundles were evident between odontoblasts and in the pre-dentin matrix (Fig. 1f). These collagen bundles extended to the distal membrane of ameloblasts (Fig. 1A,B). Mineral foci in the dentin matrix formed in close association with the collagen bundles and coalesced into the bulk of mineralized dentin (Fig. 1B,C). As the thickness of mineralized dentin expanded, a stable thickness of pre-dentin matrix was maintained by odontoblasts ( Figure S1). As dentin mineralization continued, odontoblasts moved from the DEJ further into the pulp ( Figure S1).
In contrast, the odontoblasts of Dspp −1fs/−1fs mice began to lose their polarization and cellular contacts even before dentin mineral foci coalesced into a continuous mineralized layer of dentin (Figs. 2 and S2). Pathological odontoblasts lost their organization as a sheet and showed numerous intracellular vesicles that contained cytoplasmic material at various stages of degradation (Fig. 2b, c) and swollen ER (Fig. 2f). Typical autophagosomes (Fig. 2d) and double-membraned vesicles that contained fragmented ER (Fig. 2e) indicative of active autophagy, were observed in odontoblasts. Intercellular contacts between odontoblasts were loose and irregular, exposing banded collagen fibers between odontoblasts (Fig. 2), which were less evident in WT mice (Figs. 1 and S1). Odontoblastic processes were not observed in Dspp −1fs/−1fs mice ( Figure S2). Some odontoblasts showed severe cytoplasmic pathosis, intranuclear vacuoles and nuclear destruction (Fig. 2C), which are characteristic of apoptosis. Pre-dentin deposition slowed dramatically as odontoblast cellular pathosis progressively worsened, which consequently limited dentin mineralization. Dentin mineral gradually filled the existing pre-dentin space and spread up collagen bundles between odontoblasts ( Figure S2). This ectopic mineral deposition between the odontoblasts was another sign of a disturbed mineralization process. Medium-density plaques were observed between odontoblasts and in the pre-dentin matrix (Figs. 2A,B,C,D, 2a, and S2), which corresponded to previously reported DSP-positive extracellular protein aggregates 16 .
To gain a better understanding of the nature of the odontoblast pathosis observed by electron microscopy, we conducted a series of immunohistochemical studies using light microscopy.
Loss of cellular contacts and sporadic apoptosis in the odontoblasts of Dspp −1fs mice. We used an antibody against ZO1 (zonula occludens 1), a tight junction adaptor protein, together with α-tubulin to map out the boundary of cells, to visualize and compare odontoblast intercellular contacts in Dspp +/+ (WT) and Dspp −1fs mice (Fig. 3A). In WT odontoblasts, ZO1 signal was observed in clusters along the lateral plasma membrane, but most prominently at the distal junctions. Dspp −1fs odontoblasts lacked their regular organiza- . 5000 × FIB-SEM montages from areas -2 (A), 0 (B), S1 (C), and S2 (D) from Fig  S1. (a-f). 10,000 × FIB-SEM insets of boxed areas. (A) The basement membrane separating ameloblasts from pre-dentin (orange arrows) is gradually degraded and penetrated by cell protrusions (yellow arrows). Note the polarized odontoblasts with odontoblastic processes (blue arrows) and collagen bundles (green arrows) between odontoblasts and in pre-dentin. (a) Regular and enlarged ER cisternae (white arrows) with clear membrane boundary. (b) Odontoblastic process (blue arrow) surrounded by matrix rich in collagen bundles (green arrows). (B) Dentin mineral foci (cyan arrow) in pre-dentin coalesce into a continuous layer of dentin (*). (c) Parallel ER cisternae (white arrow) and Golgi apparatus. (C) Collagen bundle spanning pre-dentin is mineralized in dentin (*). An odontoblastic process branches and extends into mineralized dentin (blue arrow   Figure S2 for area sampling locations; see Fig. 1 and S1 for WT comparisons.) (A) (area -1) Odontoblasts prior to the onset of dentin mineralization were rich in endoplasmic reticulum (ER) and mitochondria and already separated by wide intercellular spaces, exposing collagen bundles (green arrows). Medium-density protein aggregates (red arrow inset (a)) were observed in pre-dentin and extracellular vesicles (magenta arrows inset (b)) containing fragmented cisternae were evident. (B) (area 0) Pathological odontoblasts show numerous intracellular degradative vacuoles, intercellular collagen bundles (green arrow) and extracellular medium-density plaques (red arrows). Existing pre-dentin mineralizes (*) but new pre-dentin is not deposited and thins. Vacuoles (orange arrows inset (c)) contained cytoplasmic material in various stages of degradation. (C) (area S1) Apoptotic odontoblast with intranuclear vacuoles, nuclear destruction, and severe cytoplasmic pathosis. (D) (area S1) Extracellular medium-density plaques (red arrows) in pre-dentin and between odontoblasts. www.nature.com/scientificreports/ tion of tight junctions. Some odontoblasts displayed ZO1 signal on all sides of the plasma membrane, while others showed only sporadic signal on the membrane. The loss of organized tight junctions in the odontoblasts explained the loss of polarity and morphological changes in Dspp −1fs odontoblasts. After observing characteristics of apoptosis in odontoblasts using FIB-SEM, we further tested for apoptosis using multiple approaches. The activation (cleavage) of caspase 3, an executioner caspase, can be triggered by both intrinsic and extrinsic apoptosis 29 . An antibody targeting cleaved caspase 3 detected apoptosis in some Dspp −1fs odontoblasts (Fig. 3B). The TUNEL assay detects DNA fragmentation, the last phase of apoptosis. TUNEL-positive odontoblasts were also observed in the Dspp −1fs mice (Fig. 3C,D). Together, these observations demonstrated that apoptosis occurs, but suggested it is too limited to account for the extensive pathosis evident in Dspp −1fs odontoblasts. Intracellular accumulation and endoplasmic reticulum (ER) retention of DSPP in the odontoblasts and ameloblasts of Dspp P19L and Dspp −1fs mice. We previously reported the intracellular accumulation of DSPP in the odontoblasts of Dspp P19L and Dspp −1fs mice 12,16 . Here, we used immunohistochemistry to compare the intracellular accumulation of DSPP in odontoblasts and ameloblasts in D14 mandibular incisors from WT, Dspp P19L , Dspp −1fs and Dspp −/− mice (Fig. 4A). In the WT, intracellular DSP signal was detected at medium intensity in odontoblasts and low intensity in ameloblasts. In both Dspp P19L and Dspp −1fs incisors, intracellular DSPP signal was significantly elevated in both odontoblasts and ameloblasts and extended further incisally in ameloblasts relative to the WT. The DSP signal in Dspp −1fs odontoblasts appeared more concentrated in spots, while the DSP signal in Dspp P19L odontoblasts was spread out and occupied almost the whole cytoplasm. The patterns of accumulated DSPP signal highlighted the continuity of the tall sheet of columnar odontoblasts in Dspp P19L incisors and its disruption in Dspp −1fs incisors. Retention of mutant DSPP within ameloblasts beyond the presecretory stage was only observed in Dspp P19L incisors and suggested a mechanism whereby ameloblasts no longer synthesizing DSPP might still be affected by its retention.
DSPP immunohistochemistry of WT and Dspp −1fs D3 maxillary first molars using antibodies against α-tubulin clearly showed that the odontoblastic processes, so characteristic of WT odontoblasts, were completely absent in Dspp −1fs molars (Fig. 4B, green), whereas the DSPP-positive cells were low columnar cells on the pulp surface, usually polarized, with a large nucleus to cytoplasm ratio and no odontoblastic process (Fig. 4B, red). These findings were confirmed in 3-day-old maxillary 1st molars using an anti-FLAG antibody specific for the DYKD-DDDK flag-tag added to the Dspp −1fs construct ( Figure S3A).
We investigated the subcellular localization of DSPP accumulations in Dspp P19L and Dspp −1fs mice using an anti-KDEL antibody (Figs. 5 and S3B). A tetrapeptide motif Lys-Asp-Glu-Leu (KDEL) is an endoplasmic reticulum (ER) retrieval signal at the C-terminal of ER chaperone proteins 30 ; thus, this antibody is an ER marker. In WT odontoblasts and ameloblasts minimal overlap was observed between the DSP and KDEL signals in both incisors (Fig. 5A) and molars (Fig. 5B), indicating that the passage of WT-DSPP through the ER was relatively brief and WT-DSPP was efficiently synthesized and secreted by both odontoblasts and presecretory ameloblasts. The DSP and KDEL signals in Dspp P19L mice overlapped, with similar levels of overlap exhibited by odontoblasts and presecretory ameloblasts (Fig. 5A). The DSP and KDEL signals in Dspp −1fs mice also overlapped, but the DSP strongly predominated over the KDEL signal within Dspp −1fs odontoblasts indicating abundant accumulation of the -1 frameshifted DSPP protein in the ER. The DSP and KDEL signals in presecretory ameloblasts also overlapped in Dspp −1fs incisors, but the strength of the DSP signal did not predominate. These findings demonstrate that the WT DSPP protein does not accumulate in the ER, whereas the -1 frameshifted DSPP protein accumulates in the odontoblast ER to a much greater extent than does the p.P19L DSPP protein.

Intracellular retention of DMP1 and Type I Collagen in the odontoblasts of Dspp −1fs mice.
Since the -1 frameshifted DSPP protein was retained in the ER, we characterized the expression of DMP1 and type I collagen to see if their expression/secretion had been altered by Dspp −1fs expression/retention. In WT odontoblasts, intracellular DMP1 was only weakly detected at the distal end of odontoblast cell bodies (Fig. 6A). In Dspp −1fs odontoblasts, intracellular accumulation of DMP1 was observed (Fig. 6A). In the WT mice, type I collagen was most prominent between odontoblastic processes and in the pre-dentin and dentin matrix (Fig. 6B). In the Dspp −1fs mice, type I collagen was observed in the pre-dentin and dentin matrix and inside odontoblasts (Fig. 6B). Using the anti-KDEL antibody, we found that most of the type I collagen localized outside of the ER in WT odontoblasts (Fig. 6C). In comparison, most of the intracellular type I collagen signal overlapped the KDEL signal in Dspp −1fs odontoblasts (Fig. 6C), indicating the ER retention of type I collagen. The secretions of both DMP1 and type I collagen were altered in Dspp −1fs odontoblasts. P19L and Dspp −1fs mice may activate degradative pathways to cope with the intracellular/ER accumulated DSPP proteins. Ubiquitin is a molecular tag common for three degradative pathways: endocytosis, proteasome, and autophagy 24 . We tested for ubiquitin activity in the odontoblasts and ameloblasts of Dspp P19L and Dspp −1fs mice (Fig. 7). Ubiquitin was barely detectable in the odontoblasts and ameloblasts of WT and Dspp P19L mice, as well as the ameloblasts of Dspp −1fs mice (Fig. 7A). In contrast, ubiquitin signal was strong in Dspp −1fs odontoblasts, mostly overlapping with DSP signal (Fig. 7A,B), indicating that ubiquitin tagged the -1 frameshifted DSPP protein in Dspp −1fs odontoblasts.

Strong activation of ubiquitin activity in the odontoblasts of Dspp −1fs mice. Odontoblasts and ameloblasts in Dspp
Autophagy activation in the odontoblasts of Dspp −1fs mice. Since typical autophagic vacuoles were observed in the odontoblasts of Dspp −1fs mice using FIB-SEM (Fig. 2), we assessed autophagy activities molecularly. Sequestosome 1 (p62) is an autophagy adaptor that binds to ubiquitylated substrates and LC3 (microtubule   www.nature.com/scientificreports/ associated protein 1 light chain 3) on the membranes of autophagic vacuoles 31 , allowing autophagy substrates to be further processed by the autophagy-lysosome system. Like ubiquitin, the p62 signal was barely detectable in odontoblasts and ameloblasts of WT and Dspp P19L mice, and ameloblasts of Dspp −1fs mice (Fig. 8A). In contrast, p62 signal was strong in the Dspp −1fs odontoblasts (Figs. 8A, S4A). Most of the p62 signal co-localized with DSPP where the intracellular DSPP signal was highest, indicating that autophagy recognized by p62 was activated when DSPP was concentrated or aggregated in Dspp −1fs odontoblasts. Next, we tested for autophagic vacuoles using an anti-LC3B antibody (Figs. 8B, S4B). LC3B was expressed at a basal level in WT odontoblasts. However, LC3B was strongly activated in Dspp −1fs odontoblasts, and some of its signal co-localized with DSPP. Mature autophagic vacuoles fuse with lysosomes for protein degradation 32 . Lysosomes, as shown by an anti-LAMP1 antibody, were isolated vesicles distributed throughout the distal portion of WT odontoblasts ( Figure S5). LAMP1 signal showed minimal overlap with DSP signal in odontoblasts www.nature.com/scientificreports/ and ameloblasts of WT and Dspp P19L mice, as well as the ameloblasts of Dspp −1fs mice ( Figure S5A). In contrast, enlarged lysosomes at a higher intensity were observed in the Dspp −1fs odontoblasts ( Figure S5). An overlap of LAMP1 and DSP signals were observed. Sometimes, the lysosome signal was high where DSP signal was weak, presumably showing partially degraded DSPP in autolysosomes. Therefore, both the ultrastructural and molecular findings strongly support the presence of autophagy activation in Dspp −1fs odontoblasts, but not in Dspp −1fs ameloblasts or in the odontoblasts and ameloblasts of WT and Dspp P19L mice.

Discussion
Dentinogenesis imperfecta (DGI) was first documented in 1883 35,36 . With accumulating cases of autosomal dominant inherited dentin disorders, Shields et al. proposed a classification system 11 for these disorders. The dentin sialophosphoprotein (DSPP) gene was characterized in 1997 4 and determined to be the causative gene for non-syndromic DGI-II, DGI-III and dentin dysplasia type II (DD-II) 5,37,38 , all of which follow an autosomal dominant pattern of inheritance. As a growing number of disease-causing human DSPP mutations were reported, they were found to fall overwhelmingly into two categories: (1) 5' mutations that altered the amino acid sequence near the signal peptide cleavage site/amino-terminus of the secreted protein, and (2) 3' -1 frameshift mutations 6,7 in the DPP coding region. A Dspp null mouse model was generated in 2003 that displayed a severe dentin phenotype 39 , but the disease phenotype followed an autosomal recessive pattern of inheritance. The loss of only one Dspp allele did not result in a dentin phenotype. Recently, Dspp P19L and Dspp −1fs mouse models 12,16 analogous to the 5' and 3' disease-causing DSPP mutations, respectively, were characterized that exhibited dental defects inherited in a dominant pattern and were more analogous to inherited conditions in humans. In this report, we further characterized the pathological mechanisms in Dspp P19L and Dspp −1fs mice, hoping to bring insights for the development of future clinical interventions. Human DSPP 5' mutations sometimes affect both dentin and enamel formation in humans, resulting in rapid enamel attrition and increased risk of pulp exposure 6 . These mutations typically affect the first three amino acids (IPV) following the signal peptide cleavage site and alter DSPP trafficking 40 . SURF4, a cargo protein that binds to the IPV motif, prioritizes the exiting of DSPP from ER 41 . In Dspp P19L mice, malformations of dentin and enamel were observed, and were associated with the intracellular accumulation of DSPP proteins 12,18 . Here we showed that both odontoblasts and ameloblasts in the Dspp P19L mice displayed strong endoplasmic reticulum (ER) retention of DSPP proteins. Although Dspp −1fs mice also showed the accumulation of DSPP proteins in the ER, the ER retention of DSPP proteins in the Dspp P19L mice was much stronger, as indicated by the number of ameloblasts with detectable intracellular DSPP signal (Fig. 4). Interestingly, strong ER retention did not activate ubiquitin in odontoblasts or ameloblasts of Dspp P19L mice, indicating neither ERAD nor autophagy were activated. A plausible pathological mechanism for Dspp P19L odontoblasts or ameloblasts seems to be the unfolded protein response (UPR), but further investigations are warranted.
The 3' DSPP mutations, on the other hand, were exclusively -1 frameshift mutations that changed the repetitive hydrophilic/acidic amino acid sequences into repetitive hydrophobic amino acid sequences of various lengths, but always longer than the native protein 6 . Unlike the accumulated DSPP signals that distributed throughout cytoplasm in Dspp P19L mice, the intracellular DSPP in Dspp −1fs mice concentrated in spots. Considering the high level of DSPP expression and predicted hydrophobicity of the frameshifted DSPP protein in Dspp −1fs mice, it likely forms protein aggregates within the secretory pathway, which may affect the secretion of DMP1 and type I collagen in the Dspp −1fs odontoblasts. Dspp −1fs odontoblasts displayed strong ubiquitin activity and autophagy activation by both molecular and ultrastructural analyses. The autophagy activity involved partial degradation of DSPP in the ER, where the hydrophobic frameshifted protein was actively synthesized, causing ER-phagy.
Pathological mechanisms and their resulting phenotypes are multifactorial. Ameloblasts and odontoblasts are derived from dental epithelium and neural crest cells (ecto-mesenchyme), respectively 42,43 . In addition, enamel and dentin mineralize in distinct ways [44][45][46] . Dentin mineral foci first appear in pre-dentin, and associate with collagen bundles near the overlying sheet of ameloblasts. These mineral foci coalesce into a continuous layer of mineralized dentin, which gradually thickens by continued deposition of dentin mineral on the odontoblast side of dentin. Characteristic enamel mineral ribbons initiate on the tips of mineralized collagen fibers that are closely associated with the distal membrane of secretory ameloblasts and elongate in the direction that the ameloblast membrane retreats, leading to the organized appositional growth of enamel 47 . When enamel reaches full thickness, ameloblasts transition into maturation stage ameloblasts that reabsorb residual enamel matrix proteins and deposit ions on the sides of the enamel crystals that initially formed during the secretory stage 48  www.nature.com/scientificreports/ The cellular responses and their effects on biomineralization were distinct in Dspp P19L and Dspp −1fs mice. No signs of odontoblast cell death were observed in the Dspp P19L mice. Dspp P19L odontoblasts retained a significant amount of P19L-DSPP protein within their ER but continued to participate in dentin mineralization. Some of the P19L-DSPP protein was secreted 12 . Since the DSP and DPP sequences in the P19L-DSPP were minimally altered, the P19L-DSPP protein may perform its physiological activities when properly secreted. The Dspp P19L dentin and enamel phenotypes arise from reduced secretion of the P19L-DSPP protein and induced pathological changes. In contrast, the − 1fs-DSPP protein has the DPP region replaced with a highly hydrophobic C-terminal sequence that aggregates in both the cell and the pre-dentin matrix and cannot serve the physiological role of DPP. It should be remembered however, that Dspp +/− mice deposit normal dentin 39 and -2 frameshifts in the DPP coding region, which truncate the DPP domain by premature termination 10 , have never been identified as a cause of inherited dentin defects in humans. These observations support the interpretation that a loss of DPP expression from a single DSPP allele does not result in dentin malformations and that the overriding cause of the dental phenotypes in Dspp −1fs mice is abnormalities caused by the mutant protein.
Ubiquitin is a common tag for three major protein degradation pathways: ubiquitin-proteasome, autophagylysosome, and endo-lysosome systems 24 . The ubiquitin chain linkage type and the resultant three-dimensional structure may determine the selection of degradative pathways 24,49 . The status of substrate proteins may also direct the selection of pathway. The ubiquitin-proteasome system is primarily for short-lived, misfolded, and damaged proteins, while autophagy tends to eliminate large and potentially detrimental cellular components, like protein aggregates and dysfunctional organelles 50,51 . With its tendency for aggregate formation, the strong activation of autophagy in Dspp −1fs odontoblasts is not surprising.
Macroautophagy, the most studied type of autophagy (called autophagy thereafter), initiates with the formation of a phagophore, which is often observed in the vicinity of the ER 52 . It remains unclear whether phagophores arise from pre-existing membrane-bounded organelles, such as ER, Golgi apparatus, and mitochondria, or are assembled de novo in the cytosol [52][53][54] . Following the initiation and elongation of a phagophore, it engulfs ubiquitin-tagged misfolded proteins, protein aggregates, or damaged organelles, and forms a double-membrane-bounded autophagosome 26,53 . Autophagy adaptor proteins, such as p62 and NBR1, bind to ubiquitinated substrates through their UBA (ubiquitin-associated) domain, and to LC3 on the autophagosome membrane through their LIR (LC3-interacting region) domain, fostering selective autophagy 31,55 . The lysosome then fuses with an autophagosome, eliminates the inner autophagosome membrane, and acidifies the compartment environment, leading to the formation of an autolysosome 25 . Within an autolysosome, lysosomal enzymes facilitate the degradation of engulfed cytoplasmic materials 32 . Other than macroautophagy, two other types of autophagy are microautophagy and chaperone-mediated autophagy, both occurring directly on lysosomes 32,56,57 . Atg (autophagy-related) proteins govern the autophagy process 53,58 . For example, LC3 is a member of the Atg8 proteins. In Dspp −1fs odontoblasts, we observed single-membrane-bounded degradative autophagic vacuoles containing cytoplasmic materials at different degradative stages more often than double-membrane-bounded autophagic vacuoles. Specifically, we identified a total of 6 double-membrane-bounded autophagic structures from Areas -3 to S4 (annotations in Figure S2A) in Dspp −1fs odontoblasts ( Figure S7). In comparison, we did not observe any similar structures in the corresponding areas of WT odontoblasts. Strong ubiquitin, p62 and LC3B signals that co-localize with DSPP protein further confirm autophagy activation in the odontoblasts of Dspp −1fs mice molecularly.
The selective autophagy of ER, or ER-phagy, referring to the direct engulfing of ER cisternae by autophagosome, is a key ER remodeling process to alleviate ER stress 59 . The ER-phagy adaptors, mostly ER membrane proteins with LIR domain, are the key to this process 27 . FAM134B is the first identified ER-phagy receptor that possesses 2 reticulon domains to modulate ER membrane curvature 60,61 . FAM134B oligomerizes under ER stress to drive ER membrane scission for ER-phagy 62 . No apparent change in Fam134b mRNA expression is noted, but FAM134B protein congregates in Dspp −1fs odontoblasts, suggesting the oligomerization of FAM134B. Procollagens are abundantly produced and about 20% of newly synthesized type I procollagen is degraded by autophagy-lysosome system due to inefficient folding or secretion 63,64 . Misfolded type I procollagen is recognized by ER chaperone calnexin that interacts with ER-phagy adaptor FAM134B. FAM134B further binds LC3 to be captured by autophagosomes 64 . Odontoblasts produce a collagen-rich pre-dentin matrix; thus, basal levels of LC3B and FAM134B are detected in odontoblasts. In Dspp −1fs odontoblasts, the abundant −1fs-DSPP protein in the ER activates ER-phagy (indicated molecularly by LC3B co-localizing with congregated FAM134B) and ultrastructurally by double-membraned autophagic vacuoles engulfing fragmented ER cisternae. ER-phagy requires ER-resident UFMylation, a ubiquitin-like post-translational modification 34 . We detected regionally concentrated UFM1 signal in Dspp −1fs odontoblasts, which is associated with ER-phagy activity.
The dentin in Dspp P19L and Dspp −1fs mice likely represents reactionary and reparative dentin, respectively 16 . The distinction between these two types of tertiary dentin lies in the survival (reactionary) or death (reparative) of the original odontoblasts 65,66 . Cell death is morphologically classified into three types: apoptosis, autophagic cell death, and necrosis 29,[67][68][69] . With the variations of cell death being revealed, the nomenclature of cell death becomes complicated and most molecular details remain obscure 29,70 . Morphologically, apoptosis is featured by cell shrinkage, membrane blebbing, and chromatin condensation 29,69 . Apoptosis occurs by intrinsic and extrinsic pathways and relies on the BCL-2 family of proteins and caspase proteases 69,71 . Characteristic apoptotic odontoblasts were observed sporadically in the Dspp −1fs mice. Necrosis is characterized by plasma membrane rupture and disruption of organelle structure 29,69 . Autophagic cell death is featured by the autophagic vacuoles and the engagement of autophagy machinery 69 . Autophagy is mainly pro-survival. It is controversial whether autophagic cell death is merely representative of a failed survival attempt or if autophagy itself can be solely responsible for cell death independent of apoptosis and necrosis 69,[72][73][74][75] . In the case of Dspp −1fs odontoblasts, autophagic activities were detected in most cells whereas apoptotic activities were detected sporadically. Cells from sub-odontoblast layers showed the expression of DSPP and DMP1 (an early odontoblast marker) to perform odontoblast activities. www.nature.com/scientificreports/ This supports the notion that the original Dspp −1fs odontoblasts die, and odontoblast-like cells are recruited to participate in reparative dentin formation. Dspp −1fs odontoblast cell death is associated principally with autophagy and sometimes with apoptosis.
With further molecular investigations of DGI using Dspp P19L and Dspp −1fs mice, we can see a predominant ER retention in the odontoblasts and ameloblasts of the Dspp P19L mice, and strongly activated autophagy activities in the odontoblasts of Dspp −1fs mice. Better understanding of the molecular details of the odontoblast pathosis are essential for the development of therapeutic interventions for the two types of DGI. For DGI-III due to 5' DSPP mutations, the administration of chemical chaperones may push the secretion of mutant DSPP that maintains a WT DPP sequence. The alleviation of the ER retention of DSPP may reduce the stress in odontoblasts and ameloblasts thus facilitates dentin and enamel formation. The DGI-II caused by 3' -1 frameshift DSPP mutations is unlikely to be remedied, because the − 1fs-DSPP proteins is noxious to odontoblasts and the pathological changes are dramatic. Characterization of the alternative dental phenotypes of Dspp P19L and Dspp −1fs and characterization of their different pathological mechanisms strongly support the recent revisions made to the Shields classification 6 .
In addition to the mechanistic investigations of DGI, the Dspp −1fs mice may serve as a valuable tool for the study of autophagy, ER-phagy, and autophagic cell death. Pathological autophagy activities are localized to odontoblasts, minimizing concerns of adverse effects due to systemic dysregulations. Autophagy activities are high in odontoblasts and enable the investigation of molecular details of autophagic cell death in a mammalian tissue and can be explored further in the Dspp −1fs mouse model.

Methods and materials
Animal used in this study. All mice used in this study were housed in Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facilities and were treated humanely according to protocols approved by the University of Michigan and Texas A&M Institutional Animal Care and Use Committees (IACUC) and were carried out in compliance with ARRIVE guidelines. Experimental protocols were designed along University and National Institutes of Health (NIH) guidelines for the humane use of animals.
Focus ion beamed scanning electronic microscopy (FIB-SEM). FIB-SEM protocols followed were described previously in detail 45 . Briefly, seven-week-old mice for this study were anesthetized and perfused with 2.5% glutaraldehyde in 0.08 M sodium cacodylate buffer (pH7.3) with 0.05% calcium chloride. Samples were post-fixed in the same fixative for 4-6 h, then changed to 0.1 M sodium cacodylate buffer (pH7.3). The mandibles were washed several times with 0.1 M sodium cacodylate buffer, lipid-stained with 1% reduced osmium tetroxide for 2 h, dehydrated using an acetone gradient, and cured in pure epoxy. Each incisor was cut into 1-mm-thick cross-sectional slices, and those for analysis were glued onto blank epoxy stubs so that the plane of section was parallel to the long axis of the tooth. The area of starting interest for this study was the point marking the start of mineralization of dentin, designated as Area 0. Area 0 and adjacent 100 µm-wide Areas to the apical side of Area 0 (designated as Area -1, -2, -3, -4 moving more apically) and to the incisal side of Area 0 (designated as Area S1, S2, S3, S4 moving more forward into the secretory stage of amelogenesis) were imaged at various magnifications using a FEI Helios Nanolab 660 DualBeam Focused Ion Beam-Scanning Electron Microscope as described previously 45 . Histological section preparation. Heads of 3-day-old (D3) wild type (Dspp +/+ ), Dspp +/−1fs and Dspp −1fs/−1fs mice, and mandibles of 14-day-old (D14) Dspp +/+ , Dspp +/−1fs , Dspp −1fs/−1fs , Dspp +/P19L , and Dspp P19L/P19L mice were harvested, fixed in 4% PFA in PBS at 4 °C overnight. The samples were decalcified at 4 °C in 4.13% disodium ethylenediaminetetraacetic acid (EDTA, pH 7.4) with agitation for 4 days (D3 samples) or 12 days (D14 samples). The samples were then dehydrated by an ethanol series, cleared by xylene, embedded in paraffin, and sectioned at 5 μm thickness. Sections were obtained from maxillary 1st molars and mandibular incisors and placed on Fisherbrand Tissue Path Superfrost Plus Gold Microscope Slides (Fisher Scientific) for histological analysis.
TUNEL assay. TUNEL assay was performed using the TUNEL Assay kit-HRP-DAB (abcam, ab206386) following the manufacturer's protocol. Images were taken using a Nikon Eclipse TE300 microscope and photographed using a Nikon DXM1200 digital camera as described previously 16 .